Chimeric coiled coil molecules

ABSTRACT

The present application describes a fusion molecule that includes coiled coil domain, which renders the molecule soluble and provides potent activity.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation application of U.S. patentapplication Ser. No. 10/273,180, filed on Oct. 18, 2002 now U.S. Pat.No. 7,081,443, which claims the benefit of priority to U.S. ProvisionalApplication No. 60/382,541, filed May 21, 2002, the contents of whichare incorporated by reference herein in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention provides for novel method for producing novelchimeric molecules that have enhanced biological activity, and easierproduction and purification processes as compared with the molecules intheir native form. The invention also provides for nucleic acids usefulfor producing biologically active chimeric polypeptides, and the fusionpolypeptides themselves.

2. Description of the Background

Transmembrane protein kinases serve as signaling receptors for a varietyof polypeptide ligands, eliciting such diverse responses as cellsurvival, proliferation and differentiation from many cell types andtissues (van der Geer et al., 1994, Annu Rev Cell Biol. 10:251-337).Receptor tyrosine kinases (RTKs) have the ability to interact withdifferent ligands and bring about various cellular responses. One typeof RTK is tyrosine kinase with immunoglobulin and epidermal growthfactor receptor homology domains (Tie), Tie2 (Dumont et al., 1993,Oncogene 8:1293-1301; Mustonen and Alitalo, 1995, J. Cell Biol.129:895-898). Tie2 is expressed predominantly on endothelial cells,hematopoietic cells, or their embryonic precursors, and it is requiredfor normal vascular development (Sato et al., 1995, Nature 376:70-74).Functional disruption of Tie2 in transgenic mice results in embryoniclethality by day E9.5 to 10.5, with effects on the microvasculatureresulting in reduced numbers of endothelial cells, and abnormalities ofvascular morphogenesis and hematopoiesis (Sato et al., 1995, Nature376:70-74). Thus, Tie2 is critical for angiogenesis and hematopoiesisduring development.

Davis et al. discovered that Ang1 is the ligand for Tie2 (Davis et al.,1996, Cell 87:1161-1169; WO9611269) (FIG. 1). Ang1 contains 498 aminoacids, including an amino-terminal secretory signal sequence (FIG. 1).Human and mouse Ang1 are 97.6% identical. The amino-terminal region,consisting of residues 100-280, is weakly related to myosin and itsrelatives, in the regions of these proteins where they are known topossess coiled-coil quaternary structure (FIG. 1). The second region,consisting of residues 280-498, has strong similarity to thecarboxy-terminal domain of fibrinogen (FIG. 1). Ang1 is a multimer, heldtogether by coiled-coil structures and disulfide crosslinks. RecombinantAng1 is a 70-kDa (reduced condition) secreted glycoprotein that binds tothe Tie2 receptor with a Kd of approximately 3.7 nM, and inducestyrosine phosphorylation of Tie2 in endothelial cells (Davis et al.,1996, Cell 87:1161-1169).

Angiopoietin-1 (Ang1) is a specific and critical growth factor for bloodvessel formation (Davis et al., 1996, Cell 87:1161-1169; Yancopoulos etal., 2000, Nature 407:242-248). Recent studies indicate that Ang1 couldbe used for preventing vascular leakages, therapeutic vasculogenesis,and therapeutic endothelial cell survival (Thurston et al., 2000, Nat.Med. 6:460-463; Chae et al., 2000, Arterioscler. Thromb. Vasc. Biol.20:2573-2578; Kwak et al., 2000, Circulation 101:2317-2324). However,Ang1 protein is not easily available, and generation of recombinant Ang1is extremely difficult with current techniques. Multimerization of thecoiled coil domains during production of the ligand hamperedpurification.

Complementary DNAs encoding angiopoietin-2 (Ang2) were isolated bylow-stringency screening of a cDNA library by using Ang1 cDNA as a probe(Maisonpierre et al., 1997, Science 277:55-60). Ang2 contains 496 aminoacids and has a secretory signal sequence. Human and mouse Ang2 are 85%identical and approximately 60% identical to Ang1. Like Ang1, Ang2 hasan amino-terminal coiled-coil domain and a carboxy-terminalfibrinogen-like domain. Ang1 and Ang2 have similar binding affinitiesfor Tie2. Ang2 acts as an antagonist of Tie2 through inhibition ofAng1-induced phosphorylation of Tie2 (Maisonpierre et al., 1997, Science277:55-60). Mouse angiopoietin-3 (Ang3) and human angiopoietin-4 (Ang4)were identified through low stringency hybridization screening with Ang1and Ang2 cDNAs (Valenzuela et al., 1999, Proc Natl Acad. Sci.96:1904-1909). Ang3 and Ang4 are probably interspecies orthologs. Ang4phosphorylates Tie2, while Ang3 inhibits Ang1-induced phosphorylation ofTie2 (Valenzuela et al., 1999, Proc Natl Acad. Sci. 96:1904-1909).

Multimeric form of Ang1 phosphorylates Tie2. In turn, phosphorylatedTie2 interacts cytoplasmically with Grb2, Grb7, Grb14, the proteintyrosine phosphatase Shp2, and the p85 subunit of phosphatidylinositol3′-kinase (PI 3′-kinase) via their SH2 domains (Jones et al., 1999, JBiol. Chem. 274:30896-30905). Association between p85 and Tie2 resultsin PI 3′-kinase activation and subsequent induction of theserine-threonine kinase Akt. (Kontos et al., 1998, Mol Cell Biol.18:4131-4140). Ang1 induces endothelial cell survival through this PI3′-kinase/Akt signaling pathway (Kim et al., 2000, Circ Res. 86:24-29).In addition, Ang1 induces endothelial cell sprouting through theactivation of PI 3′-kinase and focal adhesion kinase (Kim et al., 2000,Circ Res. 86:952-959). Thus, Tie2, PI 3′-kinase, Akt, and focal adhesionkinase are crucial elements in the signal transduction pathway leadingto survival and migration in endothelial cells. Phosphorylated Tie2 alsointeracts with Dok-R/Dok-2, leading to activation of Dok-R/Dok-2 (Jonesand Dumont, 1998, 17:1097-1108). Phosphorylated Dok-R interacts withrasGAP, Nck, and Crk (Jones and Dumont, 1998, 17:1097-1108). Thesesignaling molecules may be involved in cell migration and proliferation,organization of the cytoskeleton, and regulation of Ras signaling.Recently, it was shown that Dok-R/Dok-2 is responsible for recruitingNck and p21-activating kinase (Pak/Pak1) to the activated receptor(Master et al., 2001, EMBO J. 20:5919-5928). Localization of thisDok-R-Nck-Pak complex to the activated Tie2 at the cellular membrane iscoincident with activation of Pak kinase (Master et al., 2001, EMBO J.20:5919-5928). This signal transduction pathway may be involved inAng1-mediated migration in endothelial cells. Signal transducers andactivators of transcription (STATs) were also found to be potentialtargets of Tie2 activation (Korpelainen et al., 1999, Oncogene, 18:1-8).Phosphorylated Tie2, in turn, activates STAT3 and STAT5 (Korpelainen etal., 1999, Oncogene, 18:1-8). Since STAT3 and/or STAT5 are known to beinvolved in the regulation of cell proliferation, differentiation,migration, and survival in many biological systems, it is possible thatsome of the Tie2 functions in endothelial cells may be controlledthrough STAT pathway.

It is known that the Tie2 receptor is expressed not only in endothelialcells but also in hematopoietic stem cells (HSCs), indicating anotherpossible role of Ang1 and Tie2 in hematopoiesis (Iwama et al., 1993,Biochem Biophys Res Commun 195:301-309). In fact, Tie2 deficient miceshow severely impaired hematopoiesis (Sato et al., 1995, Nature376:70-74). And HSCs closely adhere to endothelial cells at severalsites in the embryo. Furthermore, it has been found that HSCs produceAng1, suggesting that HSCs can promote the migration of endothelialcells and establish a hematopoietic environment (Takakura et al., 2000,Cell 102:199-209).

Ang1*, made by Regeneron Pharmaceuticals, Inc., is a recombinant versionof Ang1 that is easier to produce and purify (Maisonpierre et al., 1997,Science 277:55-60; (PCT WO 98/05779)). Ang1* contains a modifiedamino-terminus and mutated Cys²⁶⁵. The biological activity ofrecombinant Ang1 and Ang1* is similar. However, some of the sameproblems are encountered in producing Ang1* because the size of Ang1* istoo large for efficient recombinant generation.

Both native Ang1 and Ang1* require extensive, expensive andlabor-intensive purification schemes that result in relatively pooryields of recombinant protein. The need for cost-effective, simplepurification schemes for biologicals intended for clinical use cannot beover-emphasized.

U.S. Pat. No. 6,455,035 discloses a method of decreasing or inhibitingvascular permeability in a mammal by administering to the mammal a Tie-2receptor activator. However, U.S. Pat. No. '035 also discloses that thecoiled coil domain of Ang1 was deleted so that the multimerization ofthe coiled coil domains would not hamper ligand purification.

WO 00/37462 discloses a method of enhancing the biological activity ofAng1 through deletion of coiled-coil domain and insertion withFc-portion of immunoglobulin for making a tetramer form of chimericfusion Ang1 (Ang1-1FD-Fc-FD). However, WO 00/37462 discloses thatAng1-1FD-Fc-FD is equivalent to Ang1* in its ability to stimulatephosphorylation of the Tie2 receptor.

Therefore, there is a need in the art to make a modified ligand moleculethat is soluble and easily produced, while having substantially similaror greater potency as the native molecule.

SUMMARY OF THE INVENTION

The claimed invention overcomes the above-mentioned problems, andprovides for novel, biologically active, soluble forms of chimericpolypeptide ligands that bind to specific receptors on cells, includinggrowth factors belonging to angiopoietin family. Such polypeptideligands are useful in promoting or inhibiting a differential functionand/or influencing the phenotype, such as growth, survival,contractility, migration, and/or proliferation, of receptor-bearingcells such as endothelial cells, hematopoietic stem cells, andendothelial precursor cells. The invention also provides for nucleicacids encoding such polypeptide ligands, and both prokaryotic andeukaryotic expression systems for producing such polypeptide ligands.According to the invention, soluble forms of the polypeptide ligandsdescribed herein may be used to promote or inhibit biological responsesin receptor-expressing cells.

The present invention is directed to a coiled coil chimeric moleculecomprising a coiled-coil domain linked to either a receptor bindingdomain of a ligand or a ligand binding domain of a receptor, which formsa biologically active multimer, and wherein the chimeric molecule in itsnon-multimeric form is not biologically active.

Without limiting the source or structure of the coiled coil domain ofthe invention in any way, in one aspect, the coiled coil domain includesthose belonging to a protein belonging to matrix protein family,transcription factor family, growth factor family or secretory proteinfamily. Furthermore, the coiled coil domain may be of a matrix protein,in particular, cartilage matrix protein or cartilage oligomeric matrixprotein.

In the coiled coil chimeric molecule, the receptor binding domain maybind to a variety of receptors, and in particular, Tie2 or Tie1receptor. In one aspect, the receptor binding domain may be afibrinogen-like domain of angiopoietin-1, angiopoietin-2, angiopoietin-3or angiopoietin-4.

In another aspect of the invention, the coiled coil chimeric moleculemay be linked directly or indirectly to an extracellular domain of thereceptor.

In the coiled coil chimeric molecule, the ligand binding domain may bindto angiopoietin-1, angiopoietin-2, angiopoietin-3 or angiopoietin-4. Theligand binding domain may comprise extracellular domain of Tie2 receptoror Tie1 receptor. The ligand may be an agonist or antagonist. And theligand may be a cytokine, hormone or growth factor.

The ligand may be also an angiopoietin-related protein. In particular,the ligand may be angiopoietin-related protein-1 (Arp1),angiopoietin-related protein-2 (ARP2), hepaticfibrinogen/angiopoietin-related protein (HFARP). The ligand may beephrin. In particular, the ligand may be EphrinA1, EphrinA2, EphrinB1,or EphrinB2. In another aspect of the invention, the ligand may bethrombospondin. In particular, the thrombospondin may be TSP-1 or TSP-2.

In yet another aspect of the invention, with respect to the chimericmolecule, the ligand may be VEGF, PDGF, EGF, erythropoietin,interleukin, RANKL, FGF or NGF. In particular, the ligand may be VEGF-A,VEGF-B, VEGF-C, VEGF-D, VEGF-E, PDGF-A, PDGF-B, PDGF-C or PDGF-D.

In still another aspect of the invention, in the coiled coil chimericmolecule discussed above, the ligand may be angiopoietin-1,angiopoietin-2, angiopoietin-3 or angiopoietin-4. In particular, theligand may be angiopoietin-1.

The present invention is also directed to an isolated nucleic acidencoding a coiled coil chimeric molecule, which is discussed above.Further, the invention is directed to an expression vector that includesthis nucleic acid. A host cell comprising the vector is also includedwithin the purview of the invention.

The present invention also includes a soluble biologically activemultimer comprising the coiled coil chimeric molecule discussed above.In particular, the multimer may be a homomer or a homomer. If themultimer is a heteromer, then it is preferred that the coiled coildomain be heterogeneous with respect to at least one specie. Themultimer may be a dimer, trimer, tetramer, pentamer, hexamer, heptamer,octamer, nanomer or decamer and so on without limitation. Moreover, themultimer may comprise coiled coil domains, which form parallel oranti-parallel structure.

The present invention is also directed to a method of promoting cellgrowth comprising contacting a coiled coil chimeric molecule comprisinga coiled-coil domain linked to a receptor binding domain of a ligand asdescribed above, to a population of cells that express receptors thatare specific for the ligand, wherein said ligand is an agonist for saidreceptor, and wherein a multimeric form of the coiled coil chimericmolecule interacts with the receptor, which results in cell growthpromotion. In particular, in this method, without being limited to anyparticular cell type, the cells may be endothelial cells, hematopoieticcells or other cells that express each specific receptor.

In another aspect of the invention, the present invention is directed toa method of promoting cell proliferation comprising contacting a coiledcoil chimeric molecule comprising a coiled-coil domain linked to areceptor binding domain of a ligand as described above, to a populationof cells that express receptors that are specific for the ligand,wherein said ligand is an agonist for said receptor, and wherein amultimeric form of the coiled coil chimeric molecule interacts with thereceptor, which results in cell proliferation. In particular, in thismethod, without being limited to any particular cell type, the cells maybe endothelial cells, hematopoietic cells or other cells that expresseach specific receptor.

In yet another aspect of the invention, the present invention isdirected to a method of decreasing or inhibiting cell proliferationcomprising contacting a coiled coil chimeric molecule comprising acoiled-coil domain linked to a ligand binding domain of a receptor asdescribed above, to a population of cells that express ligands that arespecific for the receptor, wherein a multimeric form of the coiled coilchimeric molecule interacts with the ligand, which results in decreaseor inhibition of cell proliferation. In particular, in this method,without being limited to any particular cell type, the cells may beendothelial cells, hematopoietic cells or other cells that express eachspecific receptor.

In yet another aspect of the invention, the present invention isdirected to a method of decreasing or inhibiting ligand activitycomprising contacting a coiled coil chimeric molecule comprising acoiled-coil domain linked to a ligand binding domain of a receptor asdescribed above, to a sample comprising a ligand that is specific forthe receptor, wherein a multimeric form of the coiled coil chimericmolecule binds the ligand, which results in a decrease or inhibition ofligand activity.

In still another aspect of the invention, the present invention isdirected to a method of making a chimeric molecule comprising: (A)recombinantly combining a nucleic acid encoding a coiled-coil domainwith a nucleic acid encoding either a receptor binding region of aligand or a ligand binding region of a receptor to produce a chimericgene construct; and (B) expressing the gene construct in a host cell toproduce the chimeric molecule. These and other objects of the inventionwill be more fully understood from the following description of theinvention, the referenced drawings attached hereto and the claimsappended hereto.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will become more fully understood from thedetailed description given herein below, and the accompanying drawingswhich are given by way of illustration only, and thus are not limitativeof the present invention, and wherein;

FIG. 1 shows a schematic structure of angiopoietin-1.

FIGS. 2A-2C show nucleic acid sequence (SEQ ID NO:1) and deduced aminoacid sequence (SEQ ID NO:2) of Ang1/FD (fibrinogen-like domain of Ang1).

FIGS. 3A-3C show nucleic acid sequence (SEQ ID NO:3) and deduced aminoacid sequence (SEQ ID NO:4) of GCN4/CC-Ang1/FD (coiled-coil domain ofGCN4-Ang1/FD).

FIGS. 4A-4C show nucleic acid sequence (SEQ ID NO:5) and deduced aminoacid sequence (SEQ ID NO:6) of CMP/CC-Ang1/FD (coiled-coil domain ofCMP-Ang1/FD).

FIGS. 5A-5C show nucleic acid sequence (SEQ ID NO:7) and deduced aminoacid sequence (SEQ ID NO:8) of COMP/CC-Ang1/FD (coiled-coil domain ofCOMP-Ang1/FD).

FIG. 6 shows a schematic diagram for generation of gene constructs formultimeric chimeric-Ang1. “M” stands for multiple cloning site.

FIG. 7 shows a schematic diagram of native Ang1, GCN4-Ang1/FD,CMP-Ang1/FD, and COMP-Ang1/FD.

FIGS. 8A-8E show molecular weight analysis of recombinant native Ang1(nAng1), GCN4/CC-Ang1/FD (G/CC-A1/FD or G), CMP/CC-Ang1/FD (C/CC-A1/FDor M), and COMP/CC-Ang1/FD (CO/CC-A1/FD or 0) under reducing (R) andnon-reducing (NR) conditions. In (A-D), 100 ng of each recombinantprotein (reduced and non-reduced) is separated by 4-15% sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), andelectro-blotted to nitrocellulose membranes. The nitrocellulosemembranes were Western blotted with anti-FLAG M1 antibody, washed, andincubated with horseradish peroxidase-conjugated secondary antibody.Signals were visualized by chemiluminescent detection according to themanufacturer's protocol (Amersham Pharmacia Biotech) andchemiluminescence scanner (LAS-1000, Fuji Film, Tokyo). In (E), 500 ngof each recombinant protein (non-reduced) is separated by 7% SDS-PAGE,and stained with Coomassie blue. Molecular weight marker sizes shown(left bars with numbers (kDa); right bars without numbers) were used toestimate molecular masses.

FIG. 9 shows in vitro binding assay between recombinant native Ang1,GCN4-Ang1/FD, CMP-Ang1/FD, or COMP-Ang1/FD and soluble Tie1-Fc(sTie1-Fc, T1) and soluble Tie2-Fc (sTie2-Fc, T2). Twenty nanograms ofeach protein was incubated with 100 ng of sTie1-Fc or sTie2-Fc for 4 hr,and then, protein-A conjugated agarose beads were added. The precipitatewas separated by 10% SDS-PAGE, and electro-blotted on to nitrocellulosemembranes. The nitrocellulose membranes were Western blotted withanti-FLAG M1 antibody, washed, and incubated with horseradishperoxidase-conjugated secondary antibody. Signals were visualized bychemiluminescent detection according to the manufacturer's protocol(Amersham Pharmacia Biotech) and chemiluminescence scanner (LAS-1000,Fuji Film, Tokyo). CB, control buffer; PC, positive control of sameamount of each recombinant protein without binding.

FIGS. 10A-10D show in vitro binding assay between recombinant nativeAng1, GCN4-Ang1/FD, CMP-Ang1/FD, or COMP-Ang1/FD and immobilized solubleTie2-Fc using Biacore assay. Each recombinant protein was passed over aBiacore sensor chip that had extracellular domain of Tie2-Fc and Fcprotein covalently coupled to it, followed by dissembling in absence ofthe recombinant proteins. Concentrations of the recombinant proteins areshown in each panel. Specificity of binding was assessed by substractingFc protein binding value from Tie2-Fc binding value during themeasurement. Binding of the sensor chip is provided in resonance units(RUs). Maximal binding of each recombinant protein was observed atdifferent concentrations. Binding of (A) GCN4-Ang1/FD and (B)CMP-Ang1/FD were saturated at about 30 nM, while (C) COMP-Ang1/FD, and(D) native Ang1 binding saturation point was reached at over 125 nM. Thebinding affinity of GCN4-Ang1/FD, CMP-Ang1/FD, COMP-Ang1/FD, and nativeAng1 were estimated at 158.5 nM, 67 nM, 20.5 nM, and 7.5 nM (K_(D),dissociation constant), respectively.

FIG. 11 shows comparison of GCN4-Ang1/FD, CMP-Ang1/FD, COMP-Ang1/FD, andnative Ang1 binding characteristics at 60 nM concentration.

FIG. 12 shows comparison of native Ang1, GCN4-Ang1/FD, CMP-Ang1/FD, andCOMP-Ang1/FD in a Tie2 phosphorylation assay using human umbilicalvenous endothelial cells (HUVECs). HUVECs were incubated with controlbuffer (CB), and 200 ng of native Ang1 (N), GCN4-Ang1/FD (G),CMP-Ang1/FD (M), or COMP-Ang1/FD (O) for 10 min. The cells wereharvested in extraction buffer, and 0.5 mg of protein was used forimmunoprecipitation. Tie2 proteins in the samples wereimmunoprecipitated with anti-Tie2 antibody and collected.Immunoprecipitated samples were Western blotted withanti-phospho-tyrosine antibody (upper panel), and the membrane wasre-blotted with anti-Tie2 antibody to verify equal loading of protein ineach lane (lower panel). Results were similar in three independentexperiments. Fold: Densitometric analyses are presented as the relativeratio of phospho-Tie2 to Tie2. The relative ratio measured in CB isarbitrarily presented as 1.

FIGS. 13A-13C show comparison of native Ang1, GCN4-Ang1/FD, CMP-Ang1/FD,or COMP-Ang1/FD in Akt (Ser473) phosphorylation assay in HUVECs. In eachpanel, upper band indicates that the Western blots were probed withanti-phospho-Akt (Ser473) antibody. Lower band shows the blot that wasreprobed with anti-Akt antibody to verify equal loading of protein ineach lane. (A) HUVECs were incubated with control buffer (CB), and 200ng of native Ang1 (N), GCN4-Ang1/FD (G), CMP-Ang1/FD (M), orCOMP-Ang1/FD (O) for 15 min. (B) HUVECs were incubated with controlbuffer (CB) and indicated amount of COMP-Ang1/FD. (C) HUVECs wereincubated with 200 ng of COMP-Ang1/FD for indicated times. Aftertreatment, cell lysates were harvested. Each lane contains 50 μg oftotal protein from the cell lysates. Results were similar in threeindependent experiments. Fold: Densitometric analyses are presented asthe relative ratio of phospho-Akt (Ser472) to Akt. The relative ratiomeasured for control buffer was arbitrarily set at 1. Numbers representthe mean±S.D. from three experiments.

FIGS. 14A-14T show a comparison between native Ang1, GCN4-Ang1/FD,CMP-Ang1/FD, and COMP-Ang1/FD in survival, migration, tube formation andsprouting activities of primary cultured endothelial cells.Representative photographs are shown. Native Ang1 and CMP/CC-Ang1/FDinduced notable increase in survival, migration, tube formation andsprouting activities. Notably, COMP/CC-Ang1/FD-induced survival,migration, tube formation and sprouting activities were greater thannative Ang1- and CMP/CC-Ang1/FD-induced survival, migration, tubeformation and sprouting activities. However, GCN4/CC-Ang1/FD did notappear to exhibit any notable change in survival, migration, tubeformation or sprouting activities.

DETAILED DESCRIPTION OF THE INVENTION

In the present application, “a” and “an” are used to refer to bothsingle and a plurality of objects.

As described in greater detail below, applicants have discovered amethod of using coiled-coil domains for “multimerizing” ligands, whichenhances the biological activity of such ligands that, absent suchmultimerization, would have lower levels of biological activity. Thismethod may be used to multimerize receptor binding domains from anyligand that has improved affinity and/or increased activity (i.e.signaling ability) when they were multimerized as compared to thenon-multimerized form of the ligand.

The present invention also provides for methods of using coiled-coildomains for “multimerizing” soluble receptors, which functions to makeotherwise inactive soluble receptors biologically active, or whichenhances the biological and binding activity of receptors that, absentsuch multimerization, would have lower levels of biological and bindingactivity. This method may be used to multimerize ligand binding domainsusing any receptor, which has improved affinity and/or increasedactivity (i.e. binding) when they were multimerized as compared to thenative form of the soluble receptor.

As used herein, “agonist” refers to a ligand that binds to a receptor,which activates the receptor and stimulates physiologic activity. Forinstance, Ang1 is considered to be an agonist of Tie2 receptor.

As used herein, “antagonist” refers to a ligand that tends to nullifythe action of another ligand, as a ligand that binds to a cell receptorwithout eliciting a biological response.

As used herein, “biologically active” with regard to the ligand of thepresent invention refers to the ability of a molecule to specificallybind to and signal through a native receptor, e.g. a native Tie2receptor, or to block the ability of a native Tie receptor (e.g. Tie2)to participate in signal transduction. Thus, the (native and variant)ligands of the present invention include agonists and antagonists of anative receptor, e.g. Tie2 receptor. Preferred biological activities ofthe ligands of the present invention include the ability to induce orinhibit vascularization. The ability to induce vascularization will beuseful for the treatment of biological conditions and diseases, wherevascularization is desirable. On the other hand, the ability to inhibitor block vascularization may, for example, be useful in preventing orattenuating cell proliferation and tumor growth.

Preferred biological activities of the ligands of the present inventioninclude the ability to inhibit vascular permeability. The ability toinhibit vascular permeability will be useful for treatment of medicalconditions and diseases such as diabetic retinopathy, edema, andascites. Preferred biological activities of the ligands of the presentinvention include the ability to maintain endothelial cell integrity(including preventing apoptosis). The ability to maintain endothelialcell integrity will be useful for treatment of medical conditions anddiseases such as mannitol treatment, irradiation, and sepsis.

The biological activity of the chimeric receptor, which may be insoluble form, includes its ability to inhibit or competitively inhibitthe ligand's activity by binding to its ligand. Thus, in this way, cellproliferation may be inhibited if the ligand is an agonist for cellproliferation. Alternatively, administration of chimeric receptor mayact as an enhancer of cell proliferation if the ligand is an antagonistfor cell proliferation.

It is also contemplated that chimeric ligand and chimeric receptor belabeled with a detectable label, such as radioisotope, fluorescent tag,enzymatic tag, or a chemiluminescent tag to determine ligand-receptorbinding interaction. As such assay systems employing the chimericmolecule is also contemplated.

As used herein, “chimeric ligand”, “chimeric receptor”, “chimericpolypeptide” or “chimeric molecule” refers to the combination of coiledcoil domain and a receptor binding domain or a ligand binding domain.The resultant chimeric polypeptide is capable of forming biologicallyactive multimers, which are soluble. The coiled coil domain may bederived from any source, including any animal or mammalian protein, andin particular any human protein, and further includes those that aresynthetically made. Moreover, the coiled coil domain and the ligand orreceptor constructs may be from the same or different source. It isunderstood that the chimeric construct comprises the coiled coil domainand a receptor binding domain of a ligand or a ligand binding domain ofa receptor, and further may include other components that may beincluded so long their inclusion does not interfere with the formationof a biologically active multimer that has improved solubility, ease ofrecombinant production of the chimeric polypeptide and substantiallysimilar or greater potency as the native ligand or native solublereceptor. For example, FLAG sequence may be included for ease ofpurification, provided its inclusion does not interfere with thefunction of the chimeric molecule. The FLAG sequence also may be removedif a humanized construct is desired.

As used herein, “fragments” or “functional derivatives” refers tobiologically active amino acid sequence variants and fragments of thenative ligands or receptors of the present invention, as well ascovalent modifications, including derivatives obtained by reaction withorganic derivatizing agents, post-translational modifications,derivatives with nonproteinaceous polymers, and immunoadhesins.

As used herein, “ligand binding domain” refers to the portion of thereceptor that binds to the ligand and includes the minimal portion ofthe receptor that is necessary to bind its ligand.

As used herein, “linked” refers to direct or indirect connection betweenthe multimerizing domain and the ligand or receptor. Both a directfusion between these two domains or indirect fusion as by the domainsbeing separated by a linker or an intervening domain or element arecontemplated, so long as the activity of the chimeric fusion is present.

As used herein, “multimer” or “multimeric” refers to the joining of themultimerizing agent such as the coiled coil domain to each other to forma dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer,nanomer, decamer and so on, which may be in a parallel or anti-parallelform, through intramolecular or intermolecular bonds.

As used herein, “receptor binding domain” refers to the portion of theligand that binds to the receptor and includes the minimal portion ofthe ligand that is necessary to bind its receptor. The present inventionis based on the discovery that a multimerizing agent, such as a coiledcoil domain, which was previously perceived as a source of hindrance forisolating recombinant proteins containing them, has been found toprovide advantageous features of easy recombinant protein expression andpurification, greater solubility and greater or substantially equalpotency compared with the native protein containing the coiled coildomain.

The present invention includes a multimer forming domain. In particular,coiled coil domain is exemplified. The coiled coil domain may be anyamino acid sequence that forms a coiled coil structure. While theexemplified coiled coil domains herein are those cloned from a varietyof proteins, it is understood that various mutations and derivatizationare encompassed by the invention, so long as the resultant coiled coildomain is recognized by a person of skill in the art as a coiled coilstructure and the coiled coil domain containing chimera is capable offorming a multimer, is easily soluble, and is able to provide similar orgreater potency with respect to the native ligand or receptor.

It is further understood that in certain situations, in linking togetherthe multimerzing domain with either the receptor binding domain of theligand or ligand binding domain of the receptor, the multimerizingdomain and the binding domain may be from the same protein, or they maybe from different proteins. For instance, Ang1 coiled coil domain may belinked to its own fibrinogen-like domain in a more efficiently manner.Or, a cartilage oligomeric matrix protein (COMP) could be linked to theAng1 fibrinogen-like domain

Coiled Coil

The α-helical coiled coil is probably the most widespread subunitoligomerization motif found in proteins. Accordingly, coiled coilsfulfill a variety of different functions. In several families oftranscriptional activators, for example, short leucine zippers play animportant role in positioning the DNA-binding regions on the DNA(Ellenberger et al., 1992, Cell 71:1223-1237). Coiled coils are alsoused to form oligomers of intermediate filament proteins. Coiled-coilproteins furthermore appear to play an important role in both vesicleand viral membrane fusion (Skehel and Wiley, 1998, Cell 95:871-874). Inboth cases hydrophobic sequences, embedded in the membranes to be fused,are located at the same end of the rod-shaped complex composed of abundle of long α-helices. This molecular arrangement is believed tocause close membrane apposition as the complexes are assembled formembrane fusion.

The coiled coil is often used to control oligomerization. It is found inmany types of proteins, including transcription factors such as, but notlimited to GCN4, viral fusion peptides, SNARE complexes and certain tRNAsynthetases, among others. Very long coiled coils are found in proteinssuch as tropomyosin, intermediate filaments and spindle-pole-bodycomponents.

Coiled coils involve a number of α-helices that are supercoiled aroundeach other in a highly organized manner that associate in a parallel oran antiparallel orientation. Although dimers and trimers are the mostcommon. The helices may be from the same or from different proteins.

The coiled-coil is formed by component helices coming together to burytheir hydrophobic seams. As the hydrophobic seams twist around eachhelix, so the helices also twist to coil around each other, burying thehydrophobic seams and forming a supercoil. It is the characteristicinterdigitation of side chains between neighbouring helices, known asknobs-into-holes packing, that defines the structure as a coiled coil.The helices do not have to run in the same direction for this type ofinteraction to occur, although parallel conformation is more common.Antiparallel conformation is very rare in trimers and unknown inpentamers, but more common in intramolecular dimers, where the twohelices are often connected by a short loop.

In the extracellular space, the heterotrimeric coiled-coil proteinlaminin plays an important role in the formation of basement membranes.Other examples are the thrombospondins and cartilage oligomeric matrixprotein (COMP) in which three (thrombospondins 1 and 2) or five(thrombospondins 3, 4 and COMP) chains are connected. The molecules havea flower bouquet-like appearance, and the reason for their oligomericstructure is probably the multivalent interaction of the C-terminaldomains with cellular receptors.

GCN4

The yeast transcriptional activator GCN4 is 1 of over 30 identifiedeukaryotic proteins containing the basic region leucine zipper (bZIP)DNA-binding motif (Ellenberger et al., 1992, Cell 71:1223-1237). ThebZIP dimer is a pair of continuous alpha helices that form a parallelcoiled-coil over their carboxy-terminal 34 residues and graduallydiverge toward their amino termini to pass through the major groove ofthe DNA binding site. The coiled-coil dimerization interface is orientedalmost perpendicular to the DNA axis, giving the complex the appearanceof the letter T. bZIP contains a 4-3 heptad repeat of hydrophobic andnonpolar residues that pack together in a parallel alpha-helicalcoiled-coil (Ellenberger et al., 1992, Cell 71:1223-1237). The stabilityof the dimer results from the side-by-side packing of leucines andnonpolar residues in positions a and d of the heptad repeat, as well asa limited number of intra- and interhelical salt bridges, shown in acrystal structure of the GCN4 leucine zipper peptide (Ellenberger etal., 1992, Cell 71:1223-1237).

Cartilage Matrix Protein (CMP)

CMP (matrilin-1) was isolated from bovine tracheal cartilage as ahomotrimer of subunits of M_(r) 52,000 (Paulsson and Heinegard, 1981,Biochem J. 197:367-375), where each subunit consists of a vWFA1 module,a single EGF domain, a vWFA2 module and a coiled coil domain spanningfive heptads (Kiss et al., 1989, J. Biol. Chem. 264:8126-8134; Hauserand Paulsson, 1994, J. Biol. Chem. 269:25747-25753). Electron microscopyof purified CMP showed a bouquet-like trimer structure in which eachsubunit forms an ellipsoid emerging from a common point corresponding tothe coiled coil (Hauser and Paulsson, 1994, J. Biol. Chem.269:25747-25753). The coiled coil domain in matrilin-1 has beenextensively studied. The trimeric structure is retained after completereduction of interchain disulfide bonds under non-denaturing conditions(Hauser and Paulsson, 1994, J. Biol. Chem. 269:25747-25753).

Cartilage Oligomeric Matrix Protein (COMP)

A non-collagenous glycoprotein, COMP, was first identified in cartilage(Hedbom et al., 1992, J. Biol. Chem. 267:6132-6136). The protein is a524 kDa homopentamer of five subunits which consists of an N-terminalheptad repeat region (cc) followed by four epidermal growth factor(EGF)-like domains (EF), seven calcium-binding domains (T3) and aC-terminal globular domain (TC). According to this domain organization,COMP belongs to the family of thrombospondins. Heptad repeats(abcdefg)_(n) with preferentially hydrophobic residues at positions aand d form-helical coiled-coil domains (Cohen and Parry, 1994, Science263:488-489). Recently, the recombinant five-stranded coiled-coil domainof COMP (COMPcc) was crystallized and its structure was solved at 0.2 nmresolution (Malashkevich et al., 1996, Science 274:761-765).

The present invention also provides for a nucleic acid encoding a fusionpolypeptide wherein the fusion polypeptide comprises a first subunitcomprising at least one copy of the receptor binding domain of a ligand,the first subunit being fused to the C-terminal end of a multimerizingcomponent.

Alternatively, the present invention provides for a nucleic acidencoding a fusion polypeptide wherein the fusion polypeptide comprises afirst subunit comprising at least one copy of the receptor bindingdomain of a ligand, the first subunit being fused to the N-terminal endof a multimerizing component. In particular, the multimerizing componentmay be the coiled coil domain.

The present invention also provides for a nucleic acid encoding a fusionpolypeptide wherein the fusion polypeptide comprises a first subunitcomprising at least one copy of the ligand binding domain of a receptor,the first subunit being fused to the C-terminal end of a multimerizingcomponent.

Alternatively, the present invention provides for a nucleic acidencoding a fusion polypeptide wherein the fusion polypeptide comprises afirst subunit comprising at least one copy of the ligand binding domainof a receptor, the first subunit being fused to the N-terminal end of amultimerizing component. In particular, the multimerizing component maybe the coiled coil domain.

Also provided is an expression vector comprising a nucleic acid moleculeof the invention as described herein, wherein the nucleic acid moleculeis operatively linked to an expression control sequence. Also providedis a host-vector system for the production of a fusion polypeptide whichcomprises the expression vector of the invention which has beenintroduced into a host cell suitable for expression of the fusionpolypeptide. The suitable host cell may be a bacterial cell such as E.coli, a yeast cell, such as Pichia pastoris, an insect cell, such asSpodoptera frugiperda, or a mammalian cell, such as a COS or CHO cell.

The present invention also provides for methods of producing the fusionpolypeptides of the invention by growing cells of the host-vector systemdescribed herein, under conditions permitting production of the fusionpolypeptide and recovering the fusion polypeptide so produced. Thefusion polypeptides useful for practicing the present invention may beprepared by expression in a prokaryotic or eukaryotic expression system.

The recombinant gene may be expressed and the polypeptide purifiedutilizing any number of methods. The gene may be subcloned into abacterial expression vector, such as for example, but not by way oflimitation, pZErO.

The fusion polypeptides may be purified by any technique which allowsfor the subsequent formation of a stable, biologically active protein.For example, and not by way of limitation, the factors may be recoveredfrom cells either as soluble proteins or as inclusion bodies, from whichthey may be extracted quantitatively by 8M guanidinium hydrochloride anddialysis. In order to further purify the factors, any number ofpurification methods may be used, including but not limited toconventional ion exchange chromatography, affinity chromatography,different sugar chromatography, hydrophobic interaction chromatography,reverse phase chromatography or gel filtration.

When used herein, fusion polypeptide includes functionally equivalentmolecules in which amino acid residues are substituted for residueswithin the sequence resulting in a silent or conservative change. Forexample, one or more amino acid residues within the sequence can besubstituted by another amino acid of a similar polarity which acts as afunctional equivalent, resulting in a silent or conservative alteration.Substitutes for an amino acid within the sequence may be selected fromother members of the class to which the amino acid belongs. For example,the nonpolar (hydrophobic) amino acids include alanine, leucine,isoleucine, valine, proline, phenylalanine, tryptophan and methionine.The polar neutral amino acids include glycine, serine, threonine,cysteine, tyrosine, asparagine and glutamine. The positively charged(basic) amino acids include arginine, lysine and histidine. Thenegatively charged (acidic) amino acids include aspartic acid andglutamic acid. Also included within the scope of the invention areproteins or fragments or derivatives thereof which exhibit the same orsimilar biological activity and derivatives which are differentiallymodified during or after translation, e.g., by glycosylation,proteolytic cleavage, linkage to an antibody molecule or other cellularligand, etc.

Cells that express the fusion polypeptides of the invention aregenetically engineered to produce them by, for example, transfection,transduction, electropration, or microinjection techniques.

In addition, the present invention contemplates use of the fusionpolypeptides described herein in tagged form.

Any of the methods known to one skilled in the art for the insertion ofDNA fragments into a vector may be used to construct expression vectorsencoding the fusion polypeptides of the invention using appropriatetranscriptional/translational control signals and protein codingsequences. These methods may include in vitro recombinant DNA andsynthetic techniques and in vivo recombinations (genetic recombination).Expression of nucleic acid sequence encoding the fusion polypeptides ofthe invention may be regulated by a second nucleic acid sequence so thatthe fusion polypeptide is expressed in a host transformed with therecombinant DNA molecule. For example, expression of the fusionpolypeptides described herein may be controlled by any promoter/enhancerelement known in the art. Promoters which may be used to controlexpression of the fusion polypeptide include, but are not limited to thelong terminal repeat as described in Squinto et al., (1991, Cell65:1-20); the SV40 early promoter region (Bernoist and Chambon, 1981,Nature 290:304-310), the CMV promoter, the M-MuLV 5′ terminal repeat thepromoter contained in the 3′long terminal repeat of Rous sarcoma virus(Yamamoto, et al., 1980, Cell 22:787-797), the herpes thymidine kinasepromoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. U.S.A.78:144-1445), the regulatory sequences of the metallothionein gene(Brinster et al., 1982, Nature 296:39-42); prokaryotic expressionvectors such as the β-lactamase promoter (Villa-Kamaroff, et al., 1978,Proc. Natl. Acad. Sci. U.S.A. 75:3727-3731), or the tac promoter(DeBoer, et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:21-25), see also“Useful proteins from recombinant bacteria” in Scientific American,1980, 242:74-94; promoter elements from yeast or other fungi such as theGal 4 promoter, the ADH (alcohol dehydrogenase) promoter, PGK(phosphoglycerol kinase) promoter, alkaline phosphatase promoter, andthe following animal transcriptional control regions, which exhibittissue specificity and have been utilized in transgenic animals:elastase I gene control region which is active in pancreatic acinarcells (Swift et al., 1984, Cell 38:639-646; Ornitz et al., 1986, ColdSpring Harbor Symp. Quant. Biol. 50:399-409; MacDonald, 1987, Hepatology7:425-515); insulin gene control region which is active in pancreaticbeta cells (Hanahan, 1985, Nature 315:115-122), immunoglobulin genecontrol region which is active in lymphoid cells (Grosschedl et al.,1984, Cell 38:647-658; Adames et al., 1985, Nature 318:533-538;Alexander et al., 1987, Mol. Cell. Biol. 7:1436-1444), mouse mammarytumor virus control region which is active in testicular, breast,lymphoid and mast cells (Leder et al., 1986, Cell 45:485-495), albumingene control region which is active in liver (Pinkert et al., 1987,Genes and Devel. 1:268-276), alpha-fetoprotein gene control region whichis active in liver (Krumlauf et al., 1985, Mol. Cell. Biol. 5:1639-1648;Hammer et al., 1987, Science 235:53-58); alpha 1-antitrypsin genecontrol region which is active in the liver (Kelsey et al., 1987, Genesand Devel. 1:161-171), beta-globin gene control region which is activein myeloid cells (Mogram et al., 1985, Nature 315:338-340; Kollias etal., 1986, Cell 46:89-94); myelin basic protein gene control regionwhich is active in oligodendrocyte cells in the brain (Readhead et al.,1987, Cell 48:703-712); myosin light chain-2 gene control region whichis active in skeletal muscle (Shani, 1985, Nature 314:283-286), andgonadotropic releasing hormone gene control region which is active inthe hypothalamus (Mason et al., 1986, Science 234:1372-1378).

Thus, according to the invention, expression vectors capable of beingreplicated in a bacterial or eukaryotic host comprising nucleic acidsencoding a fusion polypeptide as described herein, and in particularmodified angiopoietin, are used to transfect the host and thereby directexpression of such nucleic acid to produce fusion polypeptides which maythen be recovered in biologically active form. As used herein, abiologically active form includes a form capable of binding to therelevant receptor and causing a differentiated function and/orinfluencing the phenotype of the cell expressing the receptor. Suchbiologically active forms would, for example, induce phosphorylation ofthe tyrosine kinase domain of Tie2 receptor, or stimulation of synthesisof cellular DNA.

Expression vectors containing the nucleic acid inserts can be identifiedby without limitation, at least three general approaches: (a) DNA-DNAhybridization, (b) presence or absence of “marker” gene functions, and(c) expression of inserted sequences. In the first approach, thepresence of foreign nucleic acids inserted in an expression vector canbe detected by DNA-DNA hybridization using probes comprising sequencesthat are homologous to an inserted nucleic acid sequences. In the secondapproach, the recombinant vector/host system can be identified andselected based upon the presence or absence of certain “marker” genefunctions (e.g., thymidine kinase activity, resistance to antibiotics,transformation phenotype, occlusion body formation in baculovirus, etc.)caused by the insertion of foreign nucleic acid sequences in the vector.For example, if an efl nucleic acid sequence is inserted within themarker gene sequence of the vector, recombinants containing the insertcan be identified by the absence of the marker gene function. In thethird approach, recombinant expression vectors can be identified byassaying the foreign nucleic acid product expressed by the recombinantconstructs. Such assays can be based, for example, on the physical orfunctional properties of the nucleic acid product of interest, forexample, by binding of a ligand to a receptor or portion thereof whichmay be tagged with, for example, a detectable antibody or portionthereof or binding to antibodies produced against the protein ofinterest or a portion thereof.

The fusion polypeptide, in particular modified angiopoietin of thepresent invention, may be expressed in the host cells transiently,constitutively or permanently.

The invention herein further provides for the development of a fusionpolypeptide as a therapeutic agent for the treatment of patientssuffering from disorders involving cells, tissues or organs whichexpress the Tie2 receptor. Such molecules may be used in a method oftreatment of the human or animal body, or in a method of diagnosis.

Because Tie2 receptor has been identified in association withendothelial cells and, blocking of agonists of the receptor such asAng-1 has been shown to prevent vascularization, applicants expect thatTie2 agonist fusion polypeptides of the invention may be useful for theinduction of vascularization in diseases or disorders where suchvascularization is indicated. Such diseases or disorders would includewound healing, ischemia and diabetes. The ligands may be tested inanimal models and used therapeutically as described for other agents,such as vascular endothelial growth factor (VEGF), which is anotherendothelial cell-specific angiogenic factor.

U.S. Pat. No. 5,332,671, as well as other studies, describe in vitro andin vivo studies that may be used to demonstrate the effect of anangiogenic factor in enhancing blood flow to ischemic myocardium,enhancing wound healing, and in other therapeutic settings whereinneoangiogenesis is desired. See also European Patent Application 0 550296 A2; Banai, et al., Circulation 89:2183-2189 (1994); Unger, et al.Am. J. Physiol. 266:H1588-H1595 (1994); and Lazarous, et al. Circulation91:145-153 (1995). According to the invention, the agonist fusionpolypeptides may be used alone or in combination with one or moreadditional pharmaceutically active compounds such as, for example, VEGFor basic fibroblast growth factor (bFGF).

Conversely, antagonists of the Tie2 receptor, such as Tie2receptorbodies or Ang-2 as described in Example 9 in WO 96/31598, havebeen shown to prevent or attenuate vascularization in certain situationsand in certain amounts. Similarly, Tie2 antagonist fusion polypeptidesof the invention would also be useful for those purposes. Theseantagonists may be used alone or in combination with other compositions,such as anti-VEGF antibodies, that have been shown to be useful intreating conditions in which the therapeutic intent is to blockangiogenesis.

In other embodiments, the Tie2 agonist fusion polypeptides of theinvention described herein may be used as hematopoietic factors. Avariety of hematopoietic factors and their receptors are involved in theproliferation and/or differentiation and/or migration of the variouscells types contained within blood. Because Tie2 receptors are expressedin early hematopoietic cells, the Tie2 ligands are expected to play acomparable role in the proliferation or differentiation or migration ofthese cells. Thus, for example, Tie2 agonist fusion polypeptidecompositions may be prepared, assayed, examined in in vitro and in vivobiological systems and used therapeutically as described in any of thefollowing: U.S. Pat. No. 4,810,643; Lee, et al., Proc. Natl. Acad. Sci.USA 82:4360-4364 (1985); Wong, et al. Science, 228:810-814 (1985);Yokota, et al. Proc. Natl. Acad. Sci (USA) 81:1070 (1984); WO 9105795;and WO 95/19985.

Accordingly, the fusion polypeptides may be used to diagnose or treatconditions in which normal hematopoiesis is suppressed, including, butnot limited to anemia, thrombocytopenia, leukopenia andgranulocytopenia. In a preferred embodiment, the fusion polypeptides maybe used to stimulate differentiation of blood cell precursors insituations where a patient has a disease, such as acquired immunedeficiency syndrome (AIDS), which is associated with reduction in normalblood cell levels, or in clinical settings in which enhancement ofhematopoietic populations is desired, such as in conjunction with bonemarrow transplant, or in the treatment of aplasia or myelosuppressioncaused by radiation, chemical treatment or chemotherapy.

The fusion polypeptides of the present invention may be used alone, orin combination with other pharmaceutically active agents such as, forexample, cytokines, neurotrophins, interleukins, etc. In a preferredembodiment, the fusion polypeptides may be used in conjunction with anyof a number of factors which are known to induce stem cell or otherhematopoietic precursor proliferation, or factors acting on later cellsin the hematopoietic pathway, including, but not limited to, hemopoieticmaturation factor, thrombopoietin, stem cell factor, erythropoietin,G-CSF, GM-CSF and so on.

In an alternative embodiment, Tie2 receptor antagonist fusionpolypeptides are used to diagnose or treat patients in which the desiredresult is inhibition of a hematopoietic pathway, such as for thetreatment of myeloproliferative or other proliferative disorders ofblood forming organs such as thrombocythemias, polycythemias andleukemias. In such embodiments, treatment may comprise use of atherapeutically effective amount of the fusion polypeptides as describedherein.

Effective doses useful for treating these or other diseases or disordersmay be determined using methods known to one skilled in the art (see,for example, Fingl, et al., The Pharmacological Basis of Therapeutics,Goodman and Gilman, eds. Macmillan Publishing Co, New York, pp. 1-46(1975). Pharmaceutical compositions for use according to the inventioninclude the fusion polypeptides described above in a pharmacologicallyacceptable liquid, solid or semi-solid carrier, linked to a carrier ortargeting molecule (e.g., antibody, hormone, growth factor, etc.) and/orincorporated into liposomes, microcapsules, and controlled releasepreparation prior to administration in vivo. For example, thepharmaceutical composition may comprise a fusion polypeptide in anaqueous solution, such as sterile water, saline, phosphate buffer ordextrose solution. Alternatively, the active agents may be comprised ina solid (e.g. wax) or semi-solid (e.g. gelatinous) formulation that maybe implanted into a patient in need of such treatment. Theadministration route may be any mode of administration known in the art,including but not limited to intravenously, intrathecally,subcutaneously, intrauterinely, by injection into involved tissue,intraarterially, intranasally, orally, or via an implanted device.

Administration may result in the distribution of the active agent of theinvention throughout the body or in a localized area. For example, insome conditions which involve distant regions of the nervous system,intravenous or intrathecal administration of agent may be desirable. Insome situations, an implant containing active agent may be placed in ornear the lesioned area. Suitable implants include, but are not limitedto, gelfoam, wax, spray, or microparticle-based implants.

The present invention also provides for pharmaceutical compositionscomprising the fusion polypeptides described herein, in apharmacologically acceptable vehicle. The compositions may beadministered systemically or locally. Any appropriate mode ofadministration known in the art may be used, incuding, but not limitedto, intravenous, intrathecal, intraarterial, intranasal, oral,subcutaneous, intraperitoneal, or by local injection or surgicalimplant. Sustained release formulations are also provided for.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent totheose skilled in the art from the foregoing description andaccompanying figures. Such modifications are intended to fall within thescope of the appended claims. The following examples are offered by wayof illustration of the present invention, and not by way of limitation.

EXAMPLES Example 1 Multimerization of Ang1 for Tie2 Phosphorylation

To determine the role of the amino-terminal region in relation to Tie2receptor, truncated Ang1 that contains only fibrinogen-like domain(amino acids 284-498) (Ang1/FD) was generated. Recombinant Ang1/FD was a˜34 kDa secreted monomer, while recombinantly produced native Ang1 was amultimer (data not shown). Interestingly, the monomeric Ang1/FD did notbind to soluble Tie2, nor did it phosphorylate Tie2. In contrast, nativeAng1 bound to Tie2 and also phosphorylated it. Thus, it appeared thatboth the amino terminus and multimerization of Ang1 could be necessaryfor Tie2 binding and activation.

To determine the role of Cys⁴¹, Cys⁵⁴ and Cys²⁶⁵ in Ang1multimerization, the amino acid 17-80 region was deleted (Ang1-D1) andCys²⁶⁵ was substituted with Ser²⁶⁵ (Ang1S265). SDS-PAGE gel analysisrevealed that recombinant Ang1-D1 was present in as a trimer andAng1S265 took on several types of multimeric forms including dimer andmonomer. These data suggest that Cys⁴¹, Cys⁵⁴ and Cys²⁶⁵ of Ang1participate in inter- and extra-molecular disulfide bond formation toform Ang1 multimers.

To determine the role of coiled-coil domains for Ang1 multimerization,we gradually deleted amino-terminal portion of Ang1. SDS-PAGE gelanalysis revealed that deletion of amino acids 17-119 (Ang1-D2), aminoacids 17-153 (Ang1-D3), and amino acids 17-212 (Ang1-D4) resulted in theformation of trimer, and dimer. In vitro binding assay with solubleTie2-Fc showed that Ang1-D2, Ang1-D3, and Ang1-D4 bound to Tie2.Interestingly, trimeric Ang 1-induced Tie2 phosphorylation was greaterthan dimeric Ang1-induced Tie2 phosphorylation. The data suggest thathigher-order multimers induce greater level of Tie2 phosphorylation. Inaddition, the second coiled-coil domain (amino acids 153-261) and linkerdomain appear to be significant factors for creating higher-ordermultimerization. Moreover, multimerization of Ang1 is essential for Tie2binding.

Example 2 Generation of Gene Constructs for Native Ang1

The full cDNA of human Ang1 was amplified from human adult heart cDNAlibrary (Clontech) by PCR for 30 cycles at an annealing temperature of52° C. using sense (5′-GTGCGGATTCACAATGACAGTTTTC-3′ (SEQ ID NO:9),including BamHI restriction enzyme site; original5′-GTGCGGCAGTACAATGACAGTTTTC-3′ (SEQ ID NO:10)) and antisense primers(5′-GCTTTCAGATATCTAAAGGTCGAAT-3′ (SEQ ID NO:11), including EcoRVrestriction enzyme site; original 5′-GCTTTCAAAAATCTAAAGGTCGAAT-3′ (SEQID NO:12)). The amplified DNA was cloned into the pCR-Blunt vector(Invitrogen) and sequence determined.

Thus obtained human Ang1 cDNA was re-subcloned into a CMVpromoter-driven mammalian cell expression vector, pcDNA3.1/Myc-His(Invitrogen), which has a DNA fragment (63 bp) encoding c-myc and a6×His tag at the 3′-terminus of the coding region as an open readingframe (CMV-Ang1-M-H).

To generate the recombinant Ang1 that has an N-terminal FLAG tag, PCRwas performed on CMV-Ang1-M-H for 25 cycles at an annealing temperatureof 60° C. using sense primer (5′-CAGAAAAGCTTGGGAGAAGATAT-3′ (SEQ IDNO:13)) and antisense primer (5′-TAGAAGGCACAGTCGAGGCTGA-3′ (SEQ IDNO:14)). The PCR products were subcloned into cloning vector pCR2.1(Invitrogen) and sequenced. The insert was cut with HindIII and PmeI,then subcloned into HindIII and EcoRV sites in pFLAG-CMV1 (Sigma). Thisvector was named ‘native Ang1’.

Example 3 Generation of Gene Constructs for Chimeric Coiled-CoilContaining Ang1

In order to generate multimeric but smaller molecular weight Ang1, theamino-terminal portion of Ang1 (261 amino-acid) was replaced with theshort coiled-coil domain of yeast transcriptional activator GCN4,cartilage matrix protein (CMP; matrilin), and cartilage oligomeric matixprotein (COMP). The coiled-coil domain of GCN4 is 31-amino acids andforms a parallel dimer. The coiled-coil domain of CMP is 43-amino acidsand forms a parallel trimer. The coiled-coil domain of COMP is 45-aminoacids and forms a parallel pentamer. Dr. Richard A. Kammerer (Departmentof Structural Biology, University of Basel) provided cDNA encodingcoiled-coil domain of yeast GCN4, chicken CMP, and rat COMP.

PCR primers for coiled-coil domain of GCN4, CMP and COMP including BglIIand BamH1 restriction enzyme sites were designed.

GCN4 BglII, Sense primer: (SEQ ID NO:15)5′-cagatcttaatgaaacagctggaagacaa-3′. GCN4 BamHI, Antisense primer: (SEQID NO: 16) 5′-ttggatccttcaccaaccagttttttcagac-3′. CMP Bg1II, Senseprimer: (SEQ ID NO:17) 5′- ccagatcttagaagaagatccgtgcgaatg-3′. CMP BamHI,Antisense primer: (SEQ ID NO:18) 5′- aaggatccgatgattttgttttccagcgc-3′.COMP BglII, Sense primer: (SEQ ID NO: 19) 5′-ccagatcttagacctagccccacagatgct-3′. COMP BamHI, Antisense primer: (SEQ IDNO:20) 5′- ttggatcctccgcaagcgtcacattccatc-3′.

PCR was performed for 30 cycles at an annealing temperature of 52° C.The PCR products were subcloned into cloning vector pZErO-2 (InVitrogen)and sequenced. It was named ‘pZErO-2CCD’.

In order to generate the secretion signal sequence of hemagglutinin andFLAG including HindIII, BamH1 and XhoI restriction enzyme sites, thefollowing sense and antisense oligonucleotides were synthesized.

SHG-FLAG HindIII-BamH1-XhoI (HBX) Sense primer:

(SEQ ID NO:21) 5′AAGCTTAAGCTTGCCACCATGAAGACGATCATCGCCCTGAGCTACATCTTCTGCCTGGTATTCGCCGACTACAAGGACGATGATGACAAGGGGATCCA CTAGTCTCGAG-3′.

SHG-FLAG XhoI-BamH1-HindIII (XBH) Antisense primer:

(SEQ ID NO:22) 5′CTCGAGACTAGTGGATCCCCTTGTCATCATCGTCCTTGTAGTCGGCGAATACCAGGCAGAAGATGTAGCTCAGGGCGATGATCGTCTTCATGGT GGCAAGCTTAAGCTT-3′.

Sense and antisense nucleotides were annealed. The annealed reactantswere ligated into the HindIII and XhoI sites of the mammalian cellexpression vector, pCDNA3.1 (InVitrogen). This vector was named‘pCDNA-Signal-FLAG’, which was incubated with BglII and BamHI. Thereleased PCR fragment was inserted into BamH1 digestedpCDNA-Signal-FLAG. This vector was named ‘pCDNA-Signal-FLAG-CC’.

PCR primers for the linker and fibrinogen domain of Ang1 (from Leu261 totermination) including BamH1 and Xho1 restriction enzyme sites weredesigned.

A1LF BamH1, Sense primer: 5′-ttggatcccttgtcaatctttgcactaaag-3′ (SEQ IDNO:23).

A1LF Xho1, Antisense primer: 5′-ttctcgagtcaaaaatctaaaggtcgaatcatc-3′(SEQ ID NO:24).

PCR was performed for 30 cycles at an annealing temperature of 52° C.The PCR products were subcloned into cloning vector pZErO-2 (InVitrogen)and sequenced. It was named ‘pZErO-2A1LF’, which was incubated withBamHI and Xho1. The released PCR fragment was inserted into BamH1 andXhoI digested pCDNA-Signal-FLAG-CC. This vector was named‘pCDNA-Signal-FLAG-CC-A1LF’. FIG. 6 shows a schematic diagram forgenerating gene constructs for the multimeric chimeric-Ang1.

As described supra, four gene constructs were made and confirmed bysequence analysis. See FIG. 7.

(1) pCDNA-Signal-FLAG-human Ang1 (native Ang1).

(2) pCDNA-Signal-FLAG-coiled-coil domain of GCN4-fibrinogen domain ofAng1 (GCN4/CC-Ang1/FD).

(3) pCDNA-Signal-FLAG-coiled-coil domain of CMP-fibrinogen domain ofAng1 (CMP/CC-Ang1/FD).

(4) pCDNA-Signal-FLAG-coiled-coil domain of COMP-fibrinogen domain ofAng1 (COMP/CC-Ang1/FD).

All of the above nucleic acid molecules were constructed by standardrecombinant DNA techniques (See e.g., Molecular Cloning, A LaboratoryManual, Sambrook, et al., Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.; Current Protocols in Molecular Biology (Eds. Ausubel, etal., Greene Publ. Assoc., Wiley-Interscience, NY), sequence-verified bystandard techniques using an ABI PRISM 3100 Genetic Analyzer (AppliedBiosystems, Foster City, Calif.) and Taq Dideoxy Terminator CycleSequencing Kit using BigDye Terminator Cycle Sequencing version 2.0(Applied Biosystems, Inc., Foster City, Calif.), ABI 373A DNA sequencerand and subcloned into the mammalian expression vector pcDNA3.1(Invitrogen, Inc.).

The bridging sequences described infra were introduced to provideconvenient restriction sites and to give flexibility to the junctionsbetween the domains, but there is no indication that there is a criticalnature to these bridging sequences, though varying the length of thelinker in some of these constructs led to some variation in the amountof protein produced.

Example 4 Construction of Native Ang1

Native Ang1 consists of a preprotrypsin leader sequence(Met-Ser-Ala-Leu-Leu-Ile-Leu-Ala-Leu-Val-Gly-Ala-Ala-Ala (SEQ ID NO:25))at its amino terminus to allow for secretion (bases 1-42), a FLAG tagsequence (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (SEQ ID NO:26), bases 43-66),a bridging amino acid Leu (bases 67-69), the coding sequence of Ang1(bases 70-1470), another bridging sequence consisting of the amino acidsAsp-Ile-Gln-His-Ser-Gly-Gly-Arg-Ser-Ser-Leu-Glu-Gly-Pro-Arg-Phe (SEQ IDNO:27) (bases 1471-1518), and the Myc epitope(Glu-Gln-Lys-Leu-Ile-Ser-Glu-Glu-Asp-Leu (SEQ ID NO:28), bases1519-1548), and a short bridging sequence consisting of amino acidsAsn-Met-His-Thr-Gly (SEQ ID NO:29) (bases 1549-1563) followed by His-Tag(His-His-His-His-His-His (SEQ ID NO:30), bases 1564-1581).

Example 5 Construction of the GCN4/CC-Ang1/FD

GCN4/CC-Ang1/FD consists of a hemagglutinin signal sequence at its aminoterminus to allow for secretion (bases 1-48 of SEQ ID NO:3 (FIG. 3A), aFLAG tag sequence (bases 49-72 of SEQ ID NO:3 (FIG. 3A)), a shortbridging sequence consisting of the amino acids Gly-Ile-Leu of SEQ IDNO:3 (FIG. 3A)), the coding sequence of GCN4 coiled-coil domain (bases82-174 of SEQ ID NO:3 (FIG. 3A)), another bridging sequence of the aminoacids Gly-Ser (bases 175-180 of SEQ ID NO:3 (FIG. 3A)), and the codingsequence for the linker region of Ang1 (bases 181-249 of SEQ ID NO:3(FIG. 3A)) followed by fibrinogen domain of Ang1 (FD) (bases 250-897 ofSEQ ID NO:3 (FIGS. 3A-3C)).

Example 6 Construction of the CMP/CC-Ang1/FD

CMP/CC-Ang1/FD consists of a hemagglutinin signal sequence at its aminoterminus to allow for secretion (bases 1-48 of SEQ ID NO:5 (FIG. 4A)), aFLAG tag sequence (bases 49-72 of SEQ ID NO:5 (FIG. 4A)), a shortbridging sequence consisting of the amino acids Gly-Ile-Leu (bases 73-81of SEQ ID NO:5 (FIG. 4A)), the coding sequence of CMP coiled-coil domain(bases 82-210 of SEQ ID NO:5 (FIG. 4A)), another bridging sequenceconsisting of the amino acids Gly-Ser (bases 211-216 of SEQ ID NO:5(FIG. 4A)), and the coding sequence for the linker region of Ang1 (bases217-285 of SEQ ID NO:5 (FIG. 4A)) followed by fibrinogen domain of Ang1(FD) (bases 286-933 of SEQ ID NO:5 (FIGS. 4A-4C)).

Example 7 Construction of the COMP/CC-Ang1/FD

COMP/CC-Ang1/FD consists of a hemagglutinin signal sequence at its aminoterminus to allow for secretion (bases 1-48 of SEQ ID NO:7 (FIG. 5A)), aFLAG tag sequence (bases 49-72 of SEQ ID NO:7 (FIG. 5A)), a shortbridging sequence consisting of the amino acids Gly-Ile-Leu (bases 73-81of SEQ ID NO:7 (FIG. 5A)), the coding sequence of COMP coiled-coildomain (bases 82-221 of SEQ ID NO:7 (FIG. 5A)), another bridgingsequence consisting of amino acids Gly-Ser (bases 222-227 of SEQ ID NO:7(FIG. 5A)), and the coding sequence for the linker region of Ang1 (bases228-296 of SEQ ID NO:7 (FIG. 5A)) followed by fibrinogen domain of Ang1(FD) (bases 250-949 of SEQ ID NO:7 (FIGS. 5A-5C)).

Example 8 Characterization of Chimeric-Ang1 Protein—Molecular WeightAnalysis

The predicted molecular weights for native Ang1, GCN4/CC-Ang1/FD,CMP/CC-Ang1/FD and COMP/CC-Ang1/FD under reduced state were determinedusing the Swiss-PROT Program. Predicted weights of FLAG-tagged nativeAng1, GCN4/CC-Ang1/FD, CMP/CC-Ang1/FD and COMP/CC-Ang1/FD are 58810.82Da, 32044.28 Da, 33330.88 Da and 33522.96 Da, respectively. There arefive N-linked glycosylation sites in native Ang1, while there is onepredicted N-linked glycosylation site in each of GCN4/CC-Ang1/FD,CMP/CC-Ang1/FD and COMP/CC-Ang1/FD. These predicted N-linkedglycosylation sites could potentially increase the molecular weight byapproximately 2500 Da/site. Thus, the predicted molecular weights fornative Ang1, GCN4/CC-Ang1/FD, CMP/CC-Ang1/FD and COMP/CC-Ang1/FD underreducing conditions are approximately, 71.3 kDa, 34.5 kDa, 35.8 kDa, and36.0 kDa, respectively. Subsequent SDS PAGE analyses (FIG. 8) of COScell-derived protein described infra confirmed these approximatemolecular weights. Molecular weights of native Ang1 (˜75 kDa),GCN4/CC-Ang1/FD (˜40 kDa), CMP/CC-Ang1/FD (˜43 kDa), and COMP/CC-Ang1/FD(˜44 kDa) were determined under reducing conditions.

Variable sizes of native Ang1 and the expected size of GCN4/CC-Ang1/FD(˜80 kDa) were shown under non-reducing conditions. However,CMP/CC-Ang1/FD (˜140 kDa and ˜180 kDa) and COMP/CC-Ang1/FD (˜180 kDa and˜250 kDa) exhibited an unexpectedly large molecular weight undernon-reducing conditions as compared with the expected molecular weightsof recombinant CMP/CC-Ang1/FD (˜135 kDa) and COMP/CC-Ang1/FD (˜220 kDa)were shown under the non-reducing condition (FIG. 8).

Example 9 Expression Level in COS Cells

Recombinant proteins of native Ang1, GCN4/CC-Ang1/FD, CMP/CC-Ang1/FD,and COMP/CC-Ang1/FD were assayed by transient expression in COS-7 cells(American Type Culture Collection, Manassas, Va.) using Effectene (aliposome) transfection method according to manufacturer's instructions(Qiagen, Inc.). Briefly, COS-7 cells were grown on gelatin-coated 100 mmdishes with Dulbecco's modified Eagle's medium (DMEM) with 10% FBS at37° C. in 5% CO₂ atmosphere. About 40-50% confluent dishes of COS-7cells were used for transfection. Effectene transfection reagents weremixed with DNA-Enhancer mixture. After 10 min incubation fortransfection-complex formation, the transfection complexes were addedonto the cells and the cells were incubated with DMEM with 5% FBS at 37°C. in 5% CO₂ atmosphere. The supernatant was harvested from transfectedcells after 48-60 hr.

Example 10 Purification of COS Cell Line Supernatant

Because the recombinant proteins contain FLAG sequence, purification isrelatively simple and straightforward using anti-FLAG M1antibody-agarose affinity gel column chromatography (Sigma-Aldrich,Inc.). Briefly, anti-FLAG M1 antibody-agarose gel was washed with 0.1 Mglycine/HCl (pH 3.5) and equilibrated with 1×TBS buffer (50 mM Tris, 150mM NaCl, pH 7.4). The supernatant containing each recombinant proteinwas passed through a column filled with anti-FLAG M1 antibody-agarosegel. After triple passage of the supernatant, the column was washed with1×TBS buffer containing 1 mM CaCl₂. The recombinant protein bound to theM1 gel was eluted with elution buffer containing 1×TBS and FLAG peptide(Sigma-Aldrich, Inc.). The relatively easy purification of therecombinant proteins provides a distinct advantage over purifying theparent protein, angiopoietin-1, which requires a highly extensive andlabor-intensive purification scheme. After purification of COS-7supernatants, recombinant proteins were quantitated using conventionalBradford method with DU 800 spectrophotometer (Beckman, Inc.) andconfirmed with Coomassie blue staining of SDS-PAGE gel. These analysesshowed that 100-200 μg of each recombinant protein/liter of COS-7 cellsupernatant was obtained, which represents moderate level of expression.

COS-7 cell supernatant yielded approximately 1 mg each of purifiednative Ang1, GCN4/CC-Ang1/FD, CMP/CC-Ang1/FD and COMP/CC-Ang1/FD thatwere used in the studies described infra to further characterize theprotein.

Example 11 Receptor Binding Analysis of COS Cell-Derived Chimeric-Ang1

Binding analysis of GCN4/CC-Ang1/FD, CMP/CC-Ang1/FD, and COMP/CC-Ang1/FDto soluble Tie2-Fc receptor was performed using in vitro binding assayand Biacore assay. In-vitro standard binding assays revealed thatGCN4/CC-Ang1/FD, CMP/CC-Ang1/FD, and COMP/CC-Ang1/FD bind to solubleTie2 receptor (FIG. 9).

Twenty nanograms of each recombinant chimeric-Ang1 protein and 100 ng ofsoluble Tie1-Fc or soluble Tie2-Fc were incubated in 500 μl Tris-buffersolution (50 mM Tris, 100 mM NaCl, pH 7.4) containing 0.02% TritonX-100at 4° C. for 2 hr. Then, 20 μl of protein-A agarose beads (Oncogene) wasadded and incubated for another 1 hr at 4° C. The protein-A conjugatedsamples were washed twice with 1 ml of Tris-buffer containing 0.02%TritonX-100. The samples were eluted with sample buffer, andheat-denatured. The samples were further separated by 10% SDS-PAGE, andelectro-blotted on to nitrocellulose membranes, and Western blotted withanti-FLAG M1 antibody to detect the bound recombinant chimeric-Ang1, andfurther washed and incubated with horseradish peroxidase-conjugatedsecondary antibody. Signal was visualized by chemiluminescent detectionaccording to the manufacturer's protocol (Amersham Pharmacia Biotech)using chemiluminescence scanner (LAS-1000, Fuji Film, Tokyo). See FIG.9. Approximately 75%, 20%, 95% or 100% of native Ang1, GCN4-Ang1/FD,CMP-Ang1/FD, and COMP-Ang1/FD, respectively, bound to sTie2-Fc. Incontrast, none of them bound to sTie1-Fc.

Alternatively, to determine whether the COS-7 cell-derived recombinantproteins of native Ang1, GCN4/CC-Ang1/FD, CMP/CC-Ang1/FD andCOMP/CC-Ang1/FD could bind to Tie-2 receptor, standard Biacore analysiswas performed by BIA2000 (BIAcore, Inc.). Briefly, 600 ng of Tie-2-Fcreceptor protein (R&D Inc), which is a fusion protein composed of theectodomain of Tie-2 receptor and Fc domain of human IgG1, wasimmobilized on a Biacore chip (Sensor Chip CM5). As a control, 600 ng ofFc protein, which had only the Fc domain of human IgG1, was alsoimmobilized on the same chip. The binding affinity was obtained bysubtracting the response value using Fc protein from the values obtainedusing the Tie-2-Fc protein.

The recombinant proteins including native Ang1, GCN4/CC-Ang1/FD,CMP/CC-Ang1/FD and COMP/CC-Ang1/FD were passed over the chip to allowbinding between Tie-2 receptor ectodomain and the recombinant proteins.The binding step was followed by a dissociation step that allowed thebound proteins to dissociate from the Tie-2 receptor ectodomain.GCN4/CC-Ang1/FD was completely dissociated from the immobilized Tie-2receptor ectodomain within 5 minutes, while CMP/CC-Ang1/FD,COMP/CC-Ang1/FD, and native Ang1 was not as easily dissociated, implyingthat there is a strong interaction between CMP/CC-Ang1/FD,COMP/CC-Ang1/FD, native Ang1 and Tie-2 receptor ectodomain. Todissociate these proteins from the receptor, various HCl solutions (pH4.0-2.0) and high salt (1M NaCl) solution were passed over the chip.However, these solutions were not able to disrupt the interactionbetween Tie-2 receptor ectodomain and the recombinant proteins,indicating that there is a strong interaction between the Tie-2 receptorectodomain and the recombinant proteins (CMP/CC-Ang1/FD,COMP/CC-Ang1/FD, and native Ang1) See FIGS. 10A-10D and 11.

In addition, association phase of native Ang1 was slower thanGCN4-Ang1/FD, CMP-Ang1/FD, COMP-Ang1/FD, implying that modification ofcoiled-coil domain of the native Ang1 increased its association constantto Tie2 receptor, and that high affinity of the native Ang1 came fromits slow dissociation.

Example 12 Tie2 Phosphorylation Assay

Human umbilical vein endothelial cells (HUVECs) were prepared from humanumbilical cords by collagenase digestion and maintained as previouslydescribed (Kim et al., 2000 Circ. Res. 86: 24-29). The primary culturedcells used for the biochemical assays were between passages 2 and 3.Primary cultured HUVECs were incubated with control buffer, and 200 ngof native Ang1, GCN4/CC-Ang1/FD, CMP/CC-Ang1/FD, or COMP/CC-Ang1/FD for10 min. The cells were harvested in extraction buffer, and 0.5 mg ofprotein was used for immunoprecipitation. Tie2 proteins in the samplewas immunoprecipitated with anti-Tie2 antibody and collected.Immunoprecipitated samples were Western blotted withanti-phospho-tyrosine antibody (upper band), and the membrane wasre-blotted with anti-Tie2 antibody (lower band) to verify equal loadingof protein in each lane. The assay revealed that COMP/CC-Ang1/FD-inducedTie2 phosphorylation (˜8.8 fold) is much higher than phosphorylationinduced by native Ang1 (˜3.6 fold) or CMP/CC-Ang1/FD (˜4.0 fold).However, GCN4/CC-Ang1/FD did not change Tie2 phosphorylation (FIG. 12).

Example 13 Akt (Ser473) Phosphorylation Assay

HUVECs were incubated with control buffer, and 200 ng of native Ang1,GCN4/CC-Ang1/FD, CMP/CC-Ang1/FD or COMP/CC-Ang1/FD for 15 min.Alternatively, HUVECs were incubated with control buffer and differentamounts of COMP/CC-Ang1/FD. HUVECs were also incubated with 200 ng ofCOMP/CC-Ang1/FD for different times. After treatment, cell lysates wereharvested. Each lane contained 50 μg of total protein from the celllysates. Western blots were probed with anti-phospho-Akt (Ser473)antibody. The blot was reprobed with anti-Akt antibody (lower bands) toverify equal loading of protein in each lane. The assay revealed thatCOMP/CC-Ang1/FD-induced Akt (Ser 473) phosphorylation (9.7 fold) isgreater than Akt (Ser 473) phosphorylation induced by native Ang1 (3.7fold) or CMP/CC-Ang1/FD (3.3 fold). However, GCN4/CC-Ang1/FD does notchange Akt (Ser 473) phosphorylation. COMP/CC-Ang1/FD-induced Akt (Ser473) phosphorylation occurs in a dose-dependent manner and persists upto 60 min (FIGS. 13A-13C, upper bands).

Example 14 Apoptosis Assay

To induce apoptosis, HUVECs were plated onto gelatinized 24-well plates(7×10⁴ cells per well) in M-199 containing 20% FBS and incubated for 12hr. The wells were extensively washed with PBS, and the medium waschanged to serum-free M-199 containing control buffer, 200 ng/ml ofnative Ang1, GCN4-Ang1/FD, CMP-Ang1/FD, or COMP-Ang1/FD, and incubatedfor 30 hr. Floating apoptotic cells were collected with 2 washes in PBS.Adherent cells were collected by trypsinization. All cells were stainedwith Annexin V FLUOS staining kit (Roche Molecular Biochemicals,Mannheim, Germany) for 15 min at 20° C. Following staining of Annexin-Vand propidium iodide (PI), the cells were analyzed on a flow cytometerand data were analyzed with CellQuest software (Becton Dickinson). Theresults were: Native Ang1 (about 47% increased cell survival),GCN4/CC-Ang1/FD (about 12% increased cell survival), CMP/CC-Ang1/FD(about 55% increased cell survival) and COMP/CC-Ang1/FD (about 71%increased cell survival). FIGS. 14A-14E. See also Table 1.

TABLE 1 Biological activities of native and chimeric Ang1 proteins inendothelial cells. Apoptosis Migration Tube length Sprouting Vector 32.4± 5.6  56.4 ± 3.5 28.4 ± 4.2 35.5 ± 7.5 Native Ang1 17.2 ± 2.3 104.7 ±4.6 42.4 ± 4.8  91.5 ± 10.5 GCN4/CC- 28.6 ± 4.3  68.0 ± 7.2 25.6 ± 3.342.5 ± 8.6 Ang1/FD CMP/CC- 14.7 ± 2.2 116.2 ± 4.2 43.6 ± 4.6 78.8 ± 8.8Ang1/FD COMP/CC-  9.4 ± 1.4 145.5 ± 6.3 55.8 ± 6.9 121.5 ± 11.5 Ang1/FD

Example 15 In Vitro Wounding Migration Assay

Cell migration assay was carried out according to Sato and Rifkin (1989,J. Cell. Biol. 109:309-315) with slight modification. Primary culturedHUVECs were grown to confluence in 30 mm diameter dish in 1 ml of normalgrowth medium. Wounds were made in the monolayer by scratching the celllayer with a double-edged razor blade and the injury line marked. Thescratch extended over an area 5-7 mm wide. After wounding, the cultureswere washed immediately with serum-free medium to remove cell debris andany soluble factors that had been released. The wounded cells werefurther incubated in serum free medium with control buffer or 200 ng/mlof native Ang1, GCN4/CC-Ang1/FD, CMP/CC-Ang1/FD, or COMP/CC-Ang1/FD.HUVECs were allowed to migrate for 10 hr and were rinsed with PBS,followed by fixing with absolute methanol and staining with Giemsa.Migration was quantitated by counting the number of cells that movedbeyond the reference line. All experiments were performed in triplicate.The results were: Native Ang1 (about 1.86 fold increase of cellmigration), GCN4/CC-Ang1/FD (about 1.20 fold increase of cellmigration), CMP/CC-Ang1/FD (about 2.06 fold increase of cell migration)and COMP/CC-Ang1/FD (about 2.58 fold increase of cell migration). SeeFIGS. 14F-14J. See also Table 1.

Example 16 Tube Formation Assay

Matrigel (Sigma-Aldrich Inc.) was thawed overnight at 4° C. and mixed tohomogeneity using cooled pipette tips. Matrigel was added to the 24-welltissue culture plate (250 μl/well) at 4° C. The 24-well plate wasbrought to a 37° C. cell culture incubator and incubated for 1 hr toallow the Matrigel to solidify. HUVECs were trypsinized, counted,resuspended in serum free M-199 medium, and added on Matrigel (1×10⁵cells/well) in the presence of control buffer, and 200 ng/ml of nativeAng1, GCN4/CC-Ang1/FD, CMP/CC-Ang1/FD or COMP/CC-Ang1/FD. Cells wereincubated for 12 hr to allow capillary-like structure to form. After 12hr, the wells were washed with PBS, fixed for 30 min in 0.5%glutaraldehyde, and the length of capillary-like tubes were quantifiedusing Image Pro-Express Software (Cyber Media). The results were: NativeAng1 (about 1.49 fold increase of tube formation), GCN4/CC-Ang1/FD(about 0.90 fold increase of tube formation), CMP/CC-Ang1/FD (about 1.53fold increase of tube formation) and COMP/CC-Ang1/FD (about 1.96 foldincrease of tube formation). See FIGS. 14K-14O. See also Table 1.

Example 17 Sprouting Assay

Cell sprouting assay in porcine pulmonary artery endothelial cells(PPAECs) was performed as previously described (Kim et al., 2000, Circ.Res. 86:952-959). Briefly, PPAECs were grown to confluence onmicrocarrier beads (diameter 175 μm; Sigma) and placed in a 2.5 mg/mlfibrinogen gel containing 2.0% heat-inactivated FBS and the indicatedrecombinant protein. Fibrin gels were incubated in DMEM with a dailyaddition of the same amount of recombinant protein. After 3 days, twoindependent investigators with no knowledge of which is the experimentalor control counted the number of sprouts using an inverted microscope.The number of endothelial sprouts with length exceeding the diameter ofthe microcarrier beads (175 μm) per 50 microcarrier beads was counted.Inter-investigator variation was <5%. The mean number from the twoinvestigators was used to estimate the number of sprout formation. Theresults were: Native Ang1 (about 2.58 fold increase of sprouting),GCN4/CC -Ang1/FD (about 1.20 fold increase of sprouting), CMP/CC-Ang1/FD(about 2.22 fold increase of sprouting) and COMP/CC-Ang1/FD (about 3.42fold increase of sprouting). See FIGS. 14P-14T. See also Table 1.

Example 18 Biophysical and Biochemical Assay

Secretion: Native Ang1, GCN4/CC-Ang1/FD, CMP/CC-Ang1/FD, andCOMP/CC-Ang1/FD are secreted from COS cells. The media and cells areharvested at 48 hr after the transfection of each gene. The proteinlevels are examined by Western-blot analysis.

Purification: Native Ang1, GCN4/CC-Ang1/FD, CMP/CC-Ang1/FD, andCOMP/CC-Ang1/FD are purified and tested for their stability, aggregationand stickiness.

All of the references cited herein are incorporated by reference intheir entirety.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention specifically described herein. Suchequivalents are intended to be encompassed in the scope of the claims.

1. A nucleic acid molecule encoding a coiled coil chimeric moleculecomprising a coiled-coil domain of cartilage oligomeric matrix proteinlinked to a Tie2 receptor binding domain which is a fibrinogen-likedomain of angiopoietin-1.
 2. An expression vector comprising the nucleicacid of claim
 1. 3. An isolated host cell comprising the vector of claim2.
 4. A method of producing a chimeric molecule comprising expressingthe nucleic acid molecule, encoding a coiled coil chimeric moleculecomprising a coiled-coil domain of cartilage oligomeric matrix proteinlinked to a Tie2 receptor binding domain which is a fibrinogen-likedomain of angiopoietin-1, in a host cell according to claim 3 to producethe chimeric molecule and recovering the chimeric molecule produced.